The present invention relates to the field of medicine, specifically to medicinal preparations affecting the immune system, and to production of such preparations.
It is known a medicinal preparation xe2x80x9csodium nucleinatexe2x80x9dxe2x80x94a sodium salt of nucleic acidxe2x80x94which is an immunological activity preparation, a white or yellowish powder easily soluble in water with formation of opalescent solutions, stimulating migration and cooperation of T-and B-lymphocytes, enhancing phagocytic activity of macrophages, and activity of nonspecific resistance factors (M. D. Mashkovsky, Medicinal Preparations, Meditsina, Moscow (1985), Vol. 2, p. 172 [in Russian]).
Injections of this preparation cause, however, pain feeling, which necessitates treatments of patients with analgesics.
The closest art for xe2x80x9csodium nucleinatexe2x80x9d is 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate, used as an immunomodulator, which also has antiinflammatory and antioxidant properties (Russian Federation Patent No. 21113222, priority: Sep. 30, 1997; IPC: A 61 K 31/04, A 61 K 31/13), being a pale-yellow crystalline powder easily soluble in water.
Administration of this preparation to patients with impaired cellular immunity, e.g., in case of malignant neoplasms, activates macrophages, interleukins and other acute-phase proteins. In case of inflammatory processes this immunomodulator inactivates macrophages for several hours, but stimulates the microbicidal system in cells.
The preparation does not cause side effects and allergic reactions, however, in patients with chronic and other diseases long-term treatment with this agent causes tolerance and decreases the efficiency of therapy with this medicinal preparation, which dictates the necessity of substituting other more efficient analogues for the preparation.
It is known a method for manufacturing the medicinal preparation including obtaining 3-amino-phthalhydrazide, its molecular rearrangement, followed by treatment with sodium hydroxide, and isolation of the target product of 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate (Russian Federation Patent No. 2138264, Priority; May 6, 1999; IPC: A 61 K 31/50, C 07 D 237/32, Bull. No. 27, Sep. 27, 1999).
This method allows to increase the yield of product and decrease the amount of waste products, however, its use is limited to manufacturing of said preparation.
The closest art to the present invention is the method for manufacturing 5-amino-2,3-dihydrophthalazine-1,4-dione (luminol) (see e.g., USSR Inventor""s Certificate No. 130903, Priority: Nov. 21, 1959; Bull. No. 16, 1960), comprising reduction of 3-nitrophtlialic acid with hydrazine hydrate in a water medium in presence of a skeletal nickel catalyst, followed by evaporation of the solution, and its heating in presence of hydrazine hydrate and acetic acid at 120xc2x0 C.
The end product of the known method is an orange-colored powder with pronounced luminescence properties, however, this compound is medically ineffective.
The object of the present invention is a medicinal preparation, whose effects is similar, but more pronounced, than those of the closest art thereof, e.g. for replacement of the known preparation in case of patient""s tolerance thereto.
The present invention the xe2x80x9cmethod for manufacturing the medicinal preparationxe2x80x9d is based on development of a procedure providing production of an efficient medicinal preparation, having immunomodulatory, antiinflammatory, and antioxidant properties.
The problem was solved by a medicinal preparation 5-amino-2,3-dihydrophthalazine-1,4-dione sodium salt having immunomodulatory, antiinflammatory, and antioxidant properties.
This problem was solved by a method for manufacturing 5-amino-2,3-dihydrophthalazine-1,4-dione sodium salt, comprising reduction of the product by hydrazine hydrate in presence of a skeletal nickel catalyst, first by interacting 3-nitro-phthalanhydride with hydrazine hydrate in acetic acid at 90-120xc2x0 C. with formation of 5-nitro-2,3-dihydrophthalazine-1,4-dione, after reduction thereof by hydrazine hydrate in a water-alkaline medium in presence of a skeletal nickel catalyst, isolating 5-amino-2,3-dihydrophthalazine-1,4-dione, which is then treated by sodium hydroxide in the presence of a lower alcohol or a ketone at 20-80xc2x0 C. to obtain the target product.
The medicinal preparation is a white or pale-yellow crystalline powder easily soluble in water.
The medicinal preparation is obtained by the following process:
3-nitro-phthalanhydride (C8H3NO6, 50-60 g) is mixed with acetic acid (CH3COOH, 120-200 ml) and heated to 90-100xc2x0 C. under mixing with dropwise admixing of hydrazine hydrate (N2H4.H2O, 15-20 ml), maintaining temperature of the reaction mixture at 105-120xc2x0 C. After addition of hydrazine hydrate, the reaction mass is boiled and held at least 20-45 min and then rapidly cooled to 70-85xc2x0 C.
Crystallized 5-nitro-2,3-dihydrophthalazine-1,4-dione (C8H5N3O4) is filtered and washed with acetic acid and distilled water. The product (5-10 g) is additionally removed from the filter, the total yield of which comprises 80-85% per 3-nitro-phthalanhydride weight.
5-nitro-2,3-dihydrophthalazine-1,4-dione (40-50 g) and potassium hydroxide (KOH, 10-15 g) are mixed in distilled water (500-700 ml) to complete dissolution. The solution is heated to 60-75xc2x0 C., hydrazine hydrate (N2H4.H2O, 12-15 ml) and Ni-Rene catalyst (2-5 g) are to the solution. This leads to a violent reaction with self-heating and emission of nitrogen (N2) and hydrogen (H2).
When temperature reaches 85-95xc2x0 C., the reaction mixture is cooled by adding distilled water. After 20-40 min, the additional catalyst (2-5 g) is fractionally added to the solution excluding the possibility of an extremely violent reaction. When self-heating is terminated, additional amounts (5-10 g) of the catalyst are added.
After completing the reaction, the solution is decanted from the precipitated catalyst, filtered, and 5-amino-2,3-dihydrophthalazine-1,4-dion (C8H7N3O2) is precipitated by acidification of the reaction mixture with an aqueous solution of hydrochloric acid (HCl) or a mixture of hydrochloric and acetic acids.
The precipitate is filtered, washed with distilled water, and dried.
The product yield per 5-nitro-2,3-dihydrophthalazine-1,4-dione weight is 82-84%.
In the final stage, 5-amino-2,3-dihydrophthalazine-1,4-dione (30-40 g) is dissolved in an aqueous solution of sodium hydroxide (10-15 g NaOH per 300-500 ml H2O) at a temperature of 20-80xc2x0 C. The solution is filtered, mixed with a lower alcohol (ROH. 1500-2000 ml), e.g., isopropyl alcohol (iso-C3H7OH) and held at 20-25xc2x0 C. for 2-3 hours, isolating the target product (C8H6N3NaO2).
Other lower alcohols or a ketone can also be used.
The target product yield per 5-amino-2,3-dihydrophthalazine-1,4-dione weight is 85-90%.
The obtained medicinal preparation is characterized by informative UV spectra in the field of 220-400 nm, taken in concentration of 20 xcexcg/ml in various solvents: water, 0.01 M solution of hydrochloric acid, 95% alcohol, and 0.1 M sodium hydroxide.
Clinical tests showed that administration of the medicinal preparation 5-amino-2,3-dihydrophthalazine-1,4-dione sodium salt to patients with impaired cellular immunity, e.g., in case of malignant neoplasms, causes activation of macrophages, which is evident by release by them of tumor necrosis factor (TNF), interleukins, and other acute-phase proteins. Besides this, the agent initiates specific reactions of T-lymphocytes.
In case of inflammatory diseases the medicinal preparation selectively (for 4-8 hours) inactivates macrophages, decreasing the contents of TNF and acute-phase proteins, that leads to attenuation of intoxication symptoms. At the same time 5-amino-2,3-dihydrophthalazine-1,4-dione sodium salt enhances super-oxidizing function and phagocytic activity of neutrophilic granulocytes, stimulating thus the microbicidal system in cells, and attenuating inflammation process.
These results are confirmed by laboratory analyses on patients, by blood tests, that characterize immunological parameters of the leukocytic and lymphocytic systems.
The medicinal preparation introduced into organism is practically completely eliminated therefrom with expired air and urine in 20-60 min. This preparation in a wide range of doses (20-1500 mg) does not cause side effects and allergic reactions, and its efficiency is similar or even higher than that of prior art immunomodulator, that allows to interchange these medicinal preparations during long-term therapy, in order to prevent the development of patient""s tolerance.
The medicinal preparation can be used in form of powder for injections or tablets for peroral administration.
Clinical efficiency of the developed medicinal preparation is confirmed by the following observations.